Analysis of FtsZ assembly by light scattering and determination of the role of divalent metal cations - PubMed (original) (raw)
Analysis of FtsZ assembly by light scattering and determination of the role of divalent metal cations
A Mukherjee et al. J Bacteriol. 1999 Feb.
Abstract
FtsZ is an ancestral homologue of tubulin that polymerizes in a GTP-dependent manner. In this study, we used 90 degrees angle light scattering to investigate FtsZ polymerization. The critical concentration for polymerization obtained by this method is similar to that obtained by centrifugation, confirming that the light scattering is proportional to polymer mass. Furthermore, the dynamics of FtsZ polymerization could be readily monitored by light scattering. Polymerization was very rapid, reaching steady state within 30 s. The length of the steady-state phase was proportional to the GTP concentration and was followed by a rapid decrease in light scattering. This decrease indicated net depolymerization that always occurred as the GTP in the reaction was consumed. FtsZ polymerization was observed over the pH range 6.5 to 7.9. Importantly, Mg2+ was not required for polymerization although it was required for the dynamic behavior of the polymers. It was reported that 7 to 25 mM Ca2+ mediated dynamic assembly of FtsZ (X. -C. Yu and W. Margolin, EMBO J. 16:5455-5463, 1997). However, we found that Ca2+ was not required for FtsZ assembly and that this concentration of Ca2+ reduced the dynamic behavior of FtsZ assembly.
Figures
FIG. 1
Assay of GTP-dependent polymerization of FtsZ by 90° angle light scattering, centrifugation, and electron microscopy. (A) Polymerization of FtsZ was assayed by 90° angle light scattering in a fluorometer. FtsZ at a concentration of 500 μg/ml (12.5 μM) in 294 μl of polymerization buffer (50 mM MES-NaOH [pH 6.5], 10 mM MgCl2, 50 mM KCl) in a fluorometer cuvette was incubated at 30°C for 8 min to establish a baseline. The reaction was initiated with the addition of nucleotide to 1 mM as indicated, and light scattering was monitored. The net change in light scattering (Δ light scattering) following nucleotide addition was plotted against time. (B) For the assay of FtsZ polymerization by centrifugation, 1 mM GDP or GTP was added to FtsZ (200 μg/ml [5 μM]) in 100 μl of polymerization buffer. The reaction mixtures were centrifuged for 15 min, and the FtsZ pellets obtained were analyzed by SDS-PAGE and Coomassie blue staining. (C and D) For analysis of FtsZ polymerization by electron microscopy, 1 mM GDP (C) or GTP (D) was added to FtsZ (200 μg/ml) in polymerization buffer at 30°C and incubated for 10 min. Samples were then prepared for visualization by electron microscopy.
FIG. 2
Light scattering is proportional to FtsZ concentration. Light scattering measurements were done as described in Materials and Methods and in the legend to Fig. 1. (A) FtsZ at different concentrations, as indicated, was incubated in polymerization buffer for 8 min at 30°C, and polymerization initiated by the addition of 1 mM GTP. (B) To obtain the critical concentration for FtsZ polymerization, the average values of the net change in light scattering (Δ light scattering) during the steady state were plotted against FtsZ concentration. The intercept on the x axis is the critical concentration.
FIG. 3
Assessment of the dynamic nature of FtsZ polymers by light scattering. Light scattering measurements were done as described in Materials and Methods and in the legend to Fig. 1. (A) FtsZ at a concentration of 500 μg/ml (12.5 μM) was incubated in polymerization buffer for 8 min at 30°C, and poly- merization was initiated by adding GTP to various final concentrations as indicated. (B) FtsZ at a concentration of 500 μg/ml (12.5 μM) was incubated for 8 min at 30°C in polymerization buffer containing different concentrations of KCl as indicated, and polymerization was initiated by adding 1 mM GTP. (C) FtsZ at a concentration of 500 μg/ml (12.5 μM) in polymerization buffer was incubated for 8 min at 30°C, and polymerization was initiated by adding 0.25 mM GTP. After depolymerization, additional rounds of FtsZ polymerization were induced by the addition of GTP as indicated.
FIG. 4
Effect of pH on FtsZ polymer formation. For polymerization at pH 6.9, 50 mM PIPES-NaOH was used; for pH 7.5 and 7.9, 50 mM HEPES-NaOH was used. The MgCl2 and KCl concentrations in these buffers were 10 and 50 mM, respectively. The standard polymerization buffer was used for the assay at pH 6.5. (A) Light scattering measurements were done as described in Materials and Methods and in the legend to Fig. 1. FtsZ at a concentration of 500 μg/ml (12.5 μM) was incubated at 30°C for 8 min in buffers at different pH values, and polymerization was initiated by adding 1 mM GTP. (B to D) For electron microscopy analyses of FtsZ polymers formed at different pHs, polymerization was initiated by adding 1 mM GTP to FtsZ at 200 μg/ml (5 μM) in buffers at pH 6.5 (B), pH 6.9 (C), and 7.5 (D). The reaction mixtures were incubated at 30°C for 10 min, and then samples were prepared for visualization by electron microscopy.
FIG. 5
Effect of Mg2+ on FtsZ polymerization and GTPase activity. Light scattering measurements were done as described in Materials and Methods and in the legend to Fig. 1. (A) FtsZ at a concentration of 500 μg/ml (12.5 μM) was incubated at 30°C for 8 min in polymerization buffer with different MgCl2 concentrations as indicated. Polymerization was initiated by adding 1 mM GTP. (B) The FtsZ GTPase reaction was initiated by adding 1 mM [γ-32P]GTP to a reaction mixture at 30°C containing FtsZ at a concentration of 500 μg/ml in polymerization buffer with different MgCl2 concentrations as indicated. At indicated times, samples were assayed for 32P formation from [γ-32P]GTP as described earlier (18). For electron microscopy analyses of FtsZ polymers formed with different concentrations of MgCl2, polymerization was initiated by adding 1 mM GTP to FtsZ at 200 μg/ml (5 μM) in polymerization buffer containing 10 mM (C), 5 mM (D), 2.5 mM (E), and 0 mM MgCl2 (F). The reaction mixtures were incubated at 30°C for 10 min, and then samples were prepared for visualization by electron microscopy.
FIG. 6
Effect of Ca2+ on FtsZ polymerization and FtsZ’s GTPase activity. (A and C) FtsZ at 500 μg/ml (12.5 μM) was incubated at 30°C for 8 min in polymerization buffer with or without 1 mM EGTA (A) or with 10 mM Ca2+ (C). Polymerization was initiated with the addition of 1 mM GTP, and light scattering measurements were done as described in Materials and Methods and in the legend to Fig. 1. (B and D) For electron microscopic analyses of FtsZ polymers, polymerization was initiated by adding 1 mM GTP to FtsZ at 200 μg/ml (5 μM) in polymerization buffer containing 1 mM EGTA (B) or 10 mM CaCl2 (D). The reaction mixtures were incubated at 30°C for 10 min, and then samples were prepared for visualization by electron microscopy. (E) The FtsZ GTPase reaction was initiated by adding 1 mM [γ-32P]GTP to a reaction mixture at 30°C containing FtsZ at a concentration of 500 μg/ml in polymerization buffer with different concentrations of CaCl2 as indicated. At indicated times, samples were assayed for 32P formation as described earlier (18, 20).
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